The present invention relates to recombinant expression vectors which have segments of deoxyribonucleic acid (DNA) that encode recombinant HIV and HCV antigens operatively linked to the sequence AGGAGGGTTTTCAT (nucleotides 1 to 15 of SEQ ID NO:1) to control expression of the antigens. These recombinant expression vectors are transformed into host cells and used in a method to express large quantities of these antigens. The invention also provides compositions containing certain of the isolated antigens, diagnostic systems containing these antigens and methods of assaying body fluids to. detect the presence of antibodies against the antigens of the invention.
The development of immunoassays for the detection of antibodies has been limited by difficulties in producing sufficient quantities of specific antigens that are essentially free of immunoreactive contaminants. The presence of contaminants that react with antibodies present in patient samples results in lower assay specificity and sensitivity and an increase in false positive results. The production of large amounts of antigen enables easier purification of antigen having a higher degree of purity and thus less immunoreactive contaminants.
The present invention overcomes the difficulties by providing a simple and highly efficient expression system that allows for the Production of large quantities of antigens. The invention relies on the efficient expression resulting from the inclusion of the nucleotide sequence AGGAGGGTTTTTCAT (which corresponds to nucleotides 1-15 of SEQ ID NO.:1) directly upstream from the ATG codon which marks the start of translation.
The invention is particularly useful for the expression of viral antigens of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV).
HIV is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). The nucleic acid sequence of the HIV proviral genome has been deduced and the location of various protein coding regions within the viral genome has been determined. Of particular interest to the present invention are the portions of the HIV genome known in the art as the gag and env regions. The gag region encodes a precursor protein that is cleaved and processed into three mature proteins, p17, p24 and p15. The HIV p24 protein has an apparent relative molecular weight of about 24,000 daltons and is known in the art as the HIV core antigen because it forms the viral capsid. Also of interest is the env region which encodes the envelope glycoproteins gp120 and gp41, which are required for viral entry into the cell. The first step in infection is the formation of a complex of gp120, gp41 and the cellular CD4 protein, binding the virus particle to the cell. The formation of this complex appears to alter the confirmation of gp41, allowing its interaction with a second cellular protein xe2x80x9cfusinxe2x80x9d, an interaction required for HIV entry into the cell.
The p24 antigen of HIV is of particular interest because studies have indicated that the first evidence of anti-HIV antibody formation (sero-conversion) in infected individuals is the appearance of antibodies induced by the p24 antigen, i.e., anti-p24 antibodies. In addition, recent studies have reported that p24 protein can be detected in blood samples even before the detection of anti p24 antibodies. Detecting the presence of either the p24 protein or anti-p24 antibodies therefore appears to be the best approach to detecting HIV infection at the earliest point in time. Furthermore, the p24 antigen reappears in the blood of infected individuals concomitant with the decline of anti-p24 antibody in patients showing the deterioration in their clinical condition that accompanies transition into full-blown AIDS. Thus, the p24 antigen can serve as an effective prognostic marker in patients undergoing therapy.
Most cases of Non-A, non-B hepatitis (NANBH) are caused by the transmissible virus now designated as hepatitis C virus (HCV). Isolates of HCV nucleic acids have been obtained and completely characterized at the sequence level. The HCV genome is comprised of a plus strand RNA molecule that codes for a single polyprotein which is cleaved to produce functionally distinct structural and nonstructural HCV proteins. Structural proteins include the capsid and envelope proteins which form the viral particle. Nonstructural proteins, such as helicase and RNA-directed RNA polymerase are required for viral function.
Some HCV gene products, or portions thereof have been expressed as fusion products. The HCV antigen C-100-3, derived from portions of the nonstructural genes designated NS3 and NS4, has been expressed as a fusion protein and used to detect anti-C-100-3 antibodies in patients with various forms of NANB hepatitis. See, for example, Kuo et al, Science, 244:362-364 (1989) and International Application No. PCT/US88/04125. A diagnostic assay based on C-100-3 antigen is commercially available from Ortho Diagnostics, Inc. (Raritan, N.J.). However, the C-100-3 antigen-based immunoassay has been reported to preferentially detect antibodies in sera from chronically infected patients. C-100-3 seroconversion generally occurs from four to six months after the onset of hepatitis, and in some cases C-100-3 fails to detect any antibody where an NANBV infection is present. Alter et al, New Eng. J. Med., 321:1538-39 (1989); Alter et al, New Eng. J. Med., 321:1494-1500 (1989); and Weiner et al, Lancet, 335:1-3 (1990). McFarlane et al, Lancet, 335:754-757 (1990), described false positive results when the C-100-3-based immunoassay was used to measure antibodies in patients with autoimmune chronic active hepatitis. In addition, Grey et al., Lancet, 335:609-610 (1990), describe false positive results using C-100-3-based immunoassay on sera from patients with liver disease caused-by a variety of conditions other than HCV. Houghton et al., U.S. Pat. No. 5,350,671, have disclosed a series of fusion proteins which include amino acids from parts of various structural and nonstructural HCV gene products fused to superoxide dismutase (SOD), many of which have no immunogenic activity when tested against HCV positive antisera.
The present invention provides compositions of recombinantly produced HIV and HCV antigens, free of bacterial and other viral components, thus enabling the detection of HIV and HCV antibodies with improved accuracy and sensitivity. The present invention also enables high yield expression of these antigens alone or as fusion proteins.
The present invention is directed to recombinant expression vectors which comprise a first nucleic acid having the sequence AGGAGGGTTTTTCAT (which corresponds to nucleotides 1-15 of SEQ ID NO.:1) operatively linked to a second nucleic acid having a sequence encoding an HIV or HCV antigen.
The preferred vectors of the inventions are pGEX7 derivatives. The pGEX7 vector contains the first nucelic acid sequence (AGGAGGGTTTTTCAT). Thus, the second nucleic acid encoding the HIV antigen or HCV antigen is operatively linked to pGEX7-derived first nucelic acid.
In addition to the recombinant expression vectors, the present invention includes host cells comprising these vectors, the recombinant HIV and HCV antigens produced by treating the host cells of the invention for a time and under conditions to cause expression of the antigen, the HIV and HCV antigens produced by this method and compositions comprising a recombinantly-produced HIV or HCV antigen of the invention. The compositions can be essentially free of procaryotic antigens or other viral-related proteins of the respective antigens.
The HIV antigen of the invention comprises three domans which are optionally joined by 1 to 5 linker amino acids. The first domain has a nucleotide sequence which encodes amino acids 1-225 of an HIV p24 antigen, the second domain has a nucleotide sequence which encodes an HIV gp41 antigen (or antigenic fragment thereof), and the third domain has a nucleotide sequence which encodes amino acids 224-232 of an HIV p24 antigen. In preferred embodiments the HIV antigen is encoded by amino acids 1-258 of SEQ ID NO:2, 4 or 6. These preferred HIV antigens are expressed from the vectors pGEXp24gp41-ANT, pGEXp24gp41-MVP and pGEXp24gp41-X84328, respectively.
The HCV antigens of the invention are the HCV capsid antigen, the HCV non-structural 794 antigen and the HCV CAP-B antigen. In preferred embodiments, the HCV capsid antigen is encoded by amino acids 1-120 from an HCV strain, and more preferably are encoded by amino acids 1-120 of SEQ ID NO:8, 10, 12 or 14. The preferred HCV capsid antigens are expressed from the vectors pGEX-C120H-V68, pGEX-C120H, pGEX-C120H-IS02 and pGEX-C120H-ISO3, respectively. In preferred embodiments the HCV non-structural 794 antigen is encoded by the amino acids of SEQ ID NO: 16 or the corresponding sequence from another HCV strain. The antigen of SEQ ID NO: 16 is preferably expressed from pGEX-NS3-794. The CAP-B antigen is encoded by the amino acids of SEQ ID NO: 18 or the corresponding sequence from another HCV strain. The antigen of SEQ ID NO: 18 is preferably expressed from pGEX-CAP-B.
Another aspect of the invention is directed to a diagnostic kit comprising an amount of a HIV antigen or HCV antigen composition of the invention sufficient to perform at least one assay.
Yet another aspect of the invention provides a method of assaying a body fluid sample for the presence of antibodies against an HIV or HCV antigen which comprises:
a) forming an immunoreaction admixture by admixing the body fluid sample with a composition of the invention;
b) maintaining the immunoreaction admixture for a time period sufficient for antibodies present against the desired antigen to immunoreact with the antigen and to form an immunoreaction product; and
c) detecting the presence of any immunoreaction product formed and thereby the presence of the desired antibodies.
The method of claim 15, wherein said detecting in step (c) can further comprise the steps of:
(i) admixing the immunoreaction product with a labeled specific binding agent to form a labeling admixture, wherein the labeled specific binding agent comprises a specific binding agent and a label;
(ii) maintaining the labeling admixture for a time period sufficient for any immunoreaction product present to bind with the labeled specific binding agent to form a labeled product; and
(iii) detecting the presence of any labeled product formed, and thereby the presence of the immunoreaction product.
In preferred embodiments, the specific binding agent can be Protein A, anti-human IgG or anti-human IgM and the label can be biotin, an enzyme, a lanthanide chelate or a radioactive isotope.
Further still, another embodiment of the invention is directed to a composition comprising the HCV capsid antigen of the invention and the HCV nonstructural 794 antigen of the invention which is essentially free of procaryotic antigens and other HCV-related proteins. These compositions can be provided as diagnostic kits and used in the methods of assaying a body fluid to detect antibodies against an HCV capsid antigen or an HCV nonstructural antigen as described above.